Contribution of human data to the analysis of human carcinogenesis

Human data strongly suggest that small doses or low concentrations of genotoxic agents cause only a relatively small number of human cancers. They emphasize the role of promotion, in particular that associated with cell proliferation. There is therefore a qualitative difference between high doses of genotoxic agents which provoke cell death and a compensatory increase in cell division, and low doses which do not. During the promotion phase, human data demonstrate the importance of induced genetic instability and defects in apoptosis as well as that of cell immortalization which play a main role for the accumulation in a cell genome of several specific lesions. Carcinogenesis is a complex process in which initial mutations do not appear to be a limiting or crucial step. This view is supported by the paramount influence of age on the induction by radiation of thyroid and breast cancer. It is also compatible with practical thresholds observed in subjects whose bones or liver were exposed to alpha-emitters, as well as with the curvilinearity in the leukemia incidence dose-response in the Japanese atomic bomb survivors. The linear no threshold model assumes that: 1) the probability of DNA lesion repair is constant whatever the dose and, hence, the number of lesions provoked in the same cell and the surrounding cells; 2) the probability for a damaged cell to evolve toward an invasive cancer is not influenced by the possible promotional effect of further irradiation or induced tissue proliferation, nor the control exerted by surrounding cells. These assumptions deserve a critical analysis.     

Variability in the longevity of Drosophila imagoes under chronic irradiation at low doses

The investigation of life span variability induced by a chronic influence of low doses gamma irradiation on the laboratory stocks of D. melanogaster, distinguishing by mobile genetic units, were carried out. Shown was the link of life span alterations in D. melanogaster with features of cytotype and genotype in tested stocks and with induced apoptotic cell death. The life span variation can be determined by a genomic destabilisation with an induction of mobile genetic elements in conditions of chronic gamma irradiation.     

The re-establishment of hypersensitive cells in the crypts of irradiated mouse intestine

Within 3-6 h of small doses of radiation (gamma-rays) the number of dead cells (apoptotic cells) in the crypts of the small intestine reaches peak values. These return to normal levels only after times later than 1 day. After higher doses elevated levels of cell death persist for longer times. The dead cells first occur most frequently at the lower positions of the crypt (median value for the distribution of apoptotic fragments is about cell position 6). At later times more dead cells are observed at higher positions. Two doses of radiation separated by various time intervals have been used to investigate when after irradiation the cell population susceptible to acute cell death is re-established. Dead cells were scored 3 or 6 h after the second dose. The yield of dead cells after two doses represents the sum of the dead cells produced by, and persisting from, the first dose and new apoptotic cells induced by the second dose. Since the temporal and dose-dependence aspects of the dead-cell yield after the first dose alone is known, the additional dead cells attributable to the second dose alone can be determined by subtraction. Within 1-2 days of small doses (0.5 Gy) the sensitive cells, recognized histologically as apoptotic cells, are re-established at the base of the crypt (around cell position 6). After higher doses (9.0 Gy) they are not re-established until about the fourth day after irradiation. Even in the enlarged regenerating crypts the sensitive cells are found at the same position at the crypt base. It has been estimated that the crypt contains five or six cells that are susceptible to low doses (0.5 Gy) (hypersensitive cells) and up to a total of only seven or eight susceptible cells that can be induced by any dose to enter the sequence of changes implicit in apoptosis. Between 4 and 10 days after an initial irradiation of 9.0 Gy the total number of susceptible cells increased from seven to eight to about 10 to 13 per crypt.     

Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.     

Studies on the mechanism of action of ionizing radiations. VII. Cellular respiration,cell division,and ionizing radiations

On x-irradiation of the eggs and sperm of Arbacia punctulata there was inhibition of respiration with relatively large doses, whereas there was an increase with small doses. The dose required to produce an increase of respiration depended on the degree of sensitivity of the cell to the effect of ionizing radiation. Sperm cells were more sensitive; then came fertilized eggs; unfertilized eggs were the least sensitive. The inhibiting effect of x-rays on cell division was observed even on irradiation with x-ray doses which produced an increase of respiration. These results are compared to similar effects produced by thiol reagents and are attributed to oxidation of the thiol compounds in the cell.     

Genetic efficacy of low doses of ionizing radiation in chronically-irradiated small mammals

Earlier we have established the genetic effects of low dose chronic irradiation in bank vole (somatic and germ cells, embryos), in pond carp (fertilized eggs, embryos, fry) and in laboratory mice (somatic and germ cells) in the range of doses from near-background to 10 cGy. These low dose effects observed in mammals and fish are not expected from extrapolation of high dose experiments. For understanding reasons this discrepancy the comparative analysis of genetic efficiency of low dose chronic irradiation and the higher doses of acute irradiation was carried out with natural populations of bank vole which inhabited the two sites differing in ground of radionuclide deposition. For comparing efficiency the linear regression model of dose-effect curve was used. Dose-effect equations were obtained for animals from two chronically irradiated bank vole populations. The mean population absorbed doses were in the range 0.04-0.68 cGy, the main part of absorbed doses consisted of external radiation of animals exposed to 137Cs gamma-rays. Dose-effect equations for acute irradiation to 137Cs gamma-rays (10-100 cGy) were determined for the same populations. Comparison of genetic efficiency was made by extrapolation, using regression coefficient beta and doubling dose estimation. For chronic exposure the doubling doses calculated from low-dose experiments are 0.1-2 cGy and the doubling doses determined from high-dose experiments are in the range of 5-20 cGy. Our hypothesis that the doubling dose estimate is calculated in higher-dose ionizing radiation experiments should be much higher than the deduced from the low dose line regression equation was verified. The doubling dose estimates for somatic cells of bank vole and those for germ cells of laboratory mice are in close agreement. The radiosensitivity of bank vole chromosomes were shown is practically the same as that for human lymphocytes since doubling dose estimates for acute irradiation close to each other. For low LET radiation a higher genetic efficiency of chronic low doses in comparison with the higher doses of acute gamma-irradiation (137Cs source) was proved by three methods.     

Study of genetic control of radiosensitivity

The results of radiation genetics studies are reviewed. The first series of studies concerned the role of heterogeneity of the human population for radiosensitivity of chromosomes in determining the pattern of dose-response relationships; correctness of extrapolation of averaged experimental data to low doses was demonstrated. In the second series of experiments, the radiation-induced adaptive response and the contribution of different factors, including genetic ones, to its formation in human cells were studied. A conclusion was made about impossibility of extrapolating data obtained for cell cultures to an organism as a whole or to a population. The third part of the study was of applied character: cytogenetic methods of biological dosimetry were used to estimate the doses of internal and external irradiation of children living on the territory of the Bryansk oblast contaminated after the Chernobyl accident. The results are discussed in the context of the present-day concepts of genetic control of sensitivity to environmental factors.     

Cytogenetic effects of extremely low doses of plutonium-238 alpha-particle irradiation in CHO K-1 cells

CHO K-1 cells were irradiated during the G1 phase with 0.5-6 rad of alpha particles. There was no appreciable cell killing in this low dose range. Significantly increased frequencies of sister-chromatid exchanges were induced by doses as low as 0.5 rad of alpha-particle irradiation, whereas increased numbers of chromosomal aberrations were observed following exposure to 2 rad. These results suggest that very low doses of alpha radiation may lead to radiation-induced genetic alterations.     

DNA damage caused by etoposide and gamma-irradiation induces gene conversion of the MHC in a mouse non-germline testis cell line

We have explored the effects of gamma-irradiation and etoposide on the gene conversion frequency between the endogenous major histocompatibility complex class II genes Abk and Ebd in a mouse testis cell line of non-germline origin with a polymerase chain reaction assay. Both gamma-rays and etoposide were shown to increase the gene conversion frequency with up to 15-fold compared to untreated cells. Etoposide, which is an agent that stabilise a cleavable complex between DNA and DNA topoisomerase II, shows an increased induction of gene conversion events with increased dose of etoposide. Cells treated with gamma-rays, which induce strand breaks, had an increased gene conversion frequency when they were subjected to low doses of irradiation, but increasing doses of irradiation did not lead to an increase of gene conversion events, which might reflect differences in the repair process depending on the extent and nature of the DNA damage. These results where DNA damage was shown to be able to induce gene conversion of endogenous genes in mouse testis cells suggests that the DNA repair system could be involved in the molecular genetic mechanism that results in gene conversion in higher eukaryotes like mammals.     

Cellular and tissue mechanisms of recovery processes in Hordeum distichum L. under irradiation

Ionizing and UV-B irradiation of barley seedlings increased the number of chromosome aberrations in vegetative and generative meristems and disturbances in microsporo- and microgametogenesis. Other irradiation-induced changes included enhanced cytomixis and an increase in the number of pathologies in meiosis and tetrads and in the polymorphism and disturbances in pollen grains. Damages induced by different irradiation types differ primarily quantitatively. The dynamics of occurrence of chromosome aberrations in the root meristem reciprocally depended on the irradiation dose. Damages induced by low UV doses were detected throughout the ontogeny of plants. Increased irradiation doses activated cytolytic processes during premeiotic interphase, meiosis, and tetrad formation, as well as after the end of gametogenesis. Exposure to maximum doses of UV radiation and moderate doses of γ-radiation restored the fertility of grain pollens. Microsporocytes, microspores, and pollen grain components undergo degeneration primarily via apoptosis. This type of cell death is apparently autonomous, being induced by the cell population itself. The results of this study confirmed the hypothesis on the positive role of cell competition in the recovery processes and adaptation of plants to mutagens.     

Effect of preliminary chronic irradiation and alpha-tocopherol on the frequency of chromosome aberrations in mouse bone marrow cells, induced by exposure to acute gamma-irradiation

The incidence of chromosome aberrations in bone marrow cells of femur did not exceed the spontaneous one in CBA mice exposed, during 70 days, to gamma-radiation at dose--rates of 33.7-35.8 nA/kg and cumulative dose of 2.75 Gy. A single acute exposure of intact animals to a dose of 2.98 Gy increased significantly the mutation level. Preirradiation with small doses increased the resistance of hereditary structures to sublethal radiation doses. Exogenous alpha-tocopherol (0.06 mg/20 g mass) protected the genetic apparatus of cells from total-body irradiation and was an additional factor decreasing the mutation level after acute exposure of mice at the background of long-term irradiation with small doses.     

Effects of irradiation on intestinal cells in vivo and in vitro

The effects of irradiation on intestinal epithelial cells were analyzed in vivo and in vitro. The in vivo study was carried out on the rat small intestine and for the in vitro study the intestinal crypt cell-line IEC-6 was used. Rat intestine and IEC-6 cells were irradiated with X-ray doses ranging between 1-16 Gy. Energy-dispersive X-ray microanalysis was used for detection of the elemental changes in the cells. Cell morphology was investigated in the scanning electron microscope, DNA-synthesis by autoradiography of 3H-thymidine incorporating nuclei and proliferation by cell counting. Our results indicate that in vivo, in the crypt cells, the increasing doses of irradiation led to increased sodium and lowered potassium and phosphorus concentrations. Corresponding ion shifts were found in the irradiated IEC-6 cells. Cells continued to proliferate up to the dose of 8 Gy, although the proliferation rate became lower with increasing dose of irradiation. The increasing dose of irradiation significantly reduced DNA-synthesis (16 Gy decreased DNA-synthesis by 50%) which resulted in a complete inhibition of cell proliferation. Analysis of goblet cells also showed characteristic radiation-dependent elemental changes. Scanning electron microscopical investigation of cells in culture revealed that most of the control cells were flat and had rather smooth cell membranes. Irradiation led to the appearance of numerous different membrane manifestations (microvilli of varying length and distribution, and blebs). Frequency of differences in the topology of the cells was related to the dose of irradiation. Our study clearly demonstrates that even low doses of irradiation cause changes in the ionic composition of the cells and inhibit DNA-synthesis and cell proliferation. The effects observed in the crypt cells in vivo were the same as in the intestinal cell line in vitro, which indicates that IEC-6 cells can be used for investigation of side effects of radiation to the abdomen.     

Evidence for reduced capacity for damage accumulation and repair in plateau-phase C3H 10T1/2 cells following multiple-dose irradiation with gamma rays

The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates.     

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.     

Cell-density dependent effects of low-dose ionizing radiation on E. coli cells

The changes in genome conformational state (GCS) induced by low-dose ionizing radiation in E. coli cells were measured by the method of anomalous viscosity time dependence (AVTD) in cellular lysates. Effects of X-rays at doses 0.1 cGy--1 Gy depended on post-irradiation time. Significant relaxation of DNA loops followed by a decrease in AVTD. The time of maximum relaxation was between 5-80 min depending on the dose of irradiation. U-shaped dose response was observed with increase of AVTD in the range of 0.1-4 Gy and decrease in AVTD at higher doses. No such increase in AVTD was seen upon irradiation of cells at the beginning of cell lysis while the AVTD decrease was the same. Significant differences in the effects of X-rays and gamma-rays at the same doses were observed suggesting a strong dependence of low-dose effects on LET. Effects of 0.01 cGy gamma-rays were studied at different cell densities during irradiation. We show that the radiation-induced changes in GCS lasted longer at higher cell density as compared to lower cell density. Only small amount of cells were hit at this dose and the data suggest cell-to-cell communication in response to low-dose ionizing radiation. This prolonged effect was also observed when cells were irradiated at high cell density and diluted to low cell density immediately after irradiation. These data suggest that cell-to-cell communication occur during irradiation or within 3 min post-irradiation. The cell-density dependent response to low-dose ionizing radiation was compared with previously reported data on exposure of E. coli cells to electromagnetic fields of extremely low frequency and extremely high frequency (millimeter waves). The body of our data show that cells can communicate in response to electromagnetic fields and ionizing radiation, presumably by reemission of secondary photons in infrared-submillimeter frequency range.     

Alteration of bacteriophage attachment capacity by near-UV irradiation.

Near-UV (NUV) (300 to 400 nm) and far-UV (FUV) (254 nm) radiations damage bacteriophage by different mechanisms. Host cell reactivation, Weigle reactivation, and multiplicity reactivation were observed upon FUV, but not upon NUV irradiation. Also, the number of his+ recombinants increased with P22 bacteriophage transduction in Salmonella typhimurium after FUV, but not after NUV irradiation. This loss of reactivation and recombination after NUV irradiation was not necessarily due to host incapability to repair phage damage. Instead, the phage genome failed to enter the host cell after NUV irradiation. In the case of NUV-irradiated T7 phage, this was determined by genetic crosses with amber mutants, which demonstrated that either "all" or "none" of a T7 genome entered the Escherichia coli cell after NUV treatment. Further studies with radioactively labeled phage indicated that irradiated phage failed to adsorb to host cells. This damage by NUV was compared with the protein-DNA cross-link observed previously, when phage particles were irradiated with NUV in the presence of H2O2. H2O2 (in nonlethal concentration) acts synergistically with NUV so that equivalent phage inactivation is achieved by much lower irradiation doses.     

Identification of druggable targets for radiation mitigation using a small interfering RNA screening assay

Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ? = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ? = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ? = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD(50/30) whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.     

The bystander effect in radiation oncogenesis: II. A quantitative model

There is strong evidence that biological response to ionizing radiation has a contribution from unirradiated "bystander" cells that respond to signals emitted by irradiated cells. We discuss here an approach incorporating a radiobiological bystander response, superimposed on a direct response due to direct energy deposition in cell nuclei. A quantitative model based on this approach is described for alpha-particle-induced in vitro oncogenic transformation. The model postulates that the oncogenic bystander response is a binary "all or nothing" phenomenon in a small sensitive subpopulation of cells, and that cells from this sensitive subpopulation are also very sensitive to direct hits from alpha particles, generally resulting in a directly hit sensitive cell being inactivated. The model is applied to recent data on in vitro oncogenic transformation produced by broad-beam or microbeam alpha-particle irradiation. Two parameters are used in analyzing the data for transformation frequency. The analysis suggests that, at least for alpha-particle-induced oncogenic transformation, bystander effects are important only at small doses-here below about 0.2 Gy. At still lower doses, bystander effects may dominate the overall response, possibly leading to an underestimation of low-dose risks extrapolated from intermediate doses, where direct effects dominate.     

Estimation of mutation rates induced by large doses of gamma, proton and neutron irradiation of the X-chromosome of the nematode Panagrellus redivivus

The radiation-resistant free-living nematode Panagrellus redivivus was used to study mutation rates in oocytes, following gamma, proton and neutron irradiation in the dose range 45-225 grays. gamma-Radiation produced approximately 0.001 lethal X-chromosomes per gray over the range tested. Proton or neutron irradiation produced approximately 0.003 lethal X-chromosomes per gray at lower doses, with the mutation rate dropping to 0.001 lethal X-chromosome per gray at the higher doses. These results suggest a dose-dependent mutation-repair system. Cell lethality was also examined. gamma-Radiation produced the greatest amount of cell lethality at all doses, while neutron irradiation had no cell lethal effect at any of the doses examined.     

Genetic effect of 3-carbethoxypsoralen, angelicin, psoralen and 8-methoxypsoralen plus 365-nm irradiation in Saccharomyces cerevisiae: induction of reversions, mitotic crossing-over, gene conversion and cytoplasmic "petite" mutations.

The genetic effects of two mono-functional photosensitizing furocoumarins, 3-carbethoxypsoralen (3-CPs) and angelicin, were compared with those of two bi-functional furocoumarins, 8-methoxypsoralen and psoralen in Saccharomyces cerevisiae. A drug concentration of 5 X 10(-5) M plus various doses of 365-nm irradiation at a dose rate of 1.2 kJ m-2 min-1 were used. Per dose of 365-nm irradiation, the frequency of induced nuclear events such as gene mutation and mitotic recombination (conversion and crossing-over) is higher for the bi-functional than for the mono-functional compounds. The higher efficiency of the bi-functional furocoumarins is also evident when the frequency of mutants is expressed as a function of survival. However, the photo-addition of the 4 furocoumarins studied leads to the same response for the induction of recombinational events per viable cell. Amongst genetically altered colonies induced in the diploid strains D5 and D7, the colonies corresponding to the induction of crossing-over are effectively produced by bi-functional furocoumarins, but are rare (D7) or even absent (D5) after treatment with monofunctional furocoumarins. This suggests a certain specificity of genetic alterations produced by the bi-functional agents. 3-CPs is the most effective inducer on the cytoplasmic "petite" mutation in stationary phase cells per unit irradiation dose or per viable cell.